Advances in Instrumentation and Software For Pathway Informed Multi-Omics Analysis Agilent Technologies

1 Yeast Omics

Biology Is Integrated

DNA RNA Protein Metabolite

2 Yeast Omics

Integrating Biological Analysis Using Pathways

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R

R

Protein B

Protein A

Protein X

HO

Sources • WikiPathways • BioCyc/MetaCyc • Generalized BioPax • KEGG

Platforms • GeneSpring • Mass Profiler Professional

• Pathway Architect

Integrating Biological Analysis Using Pathways

R

R

HO

Protein B

Protein A

Protein X

R

R

HO

Protein B

Protein A

Protein X R

R

HO

Protein B

Protein A

Protein X

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Integrating Biological Analysis Using Pathways

R

R

HO

Protein B

Protein A

Protein X

• Identifies why the pathway is active • Suggests follow-on experiments

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Baker’s Yeast is an Ideal Model Organism for Studying Pathways

• Saccharomyces cerevisiae is a widely used model organism

• Biochemistry and pathways are extensively studied

• Fully sequenced genome

• Ideal for “multi-omics” studies with the goal of facilitating research for other organisms.

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Yeast Multi-omics: Metabolomics and Proteomics

Treatments:

• Wild type (WT) - no treatment

• Calcium control (CC) - CaCl2

• FK - FK506 and CaCl2

• CA - Cyclosporin A and CaCl2

Metabolomics: Wet mill with 5:3:3 CHCl3:CH3OH:H2O. Only the aqueous is analyzed

Proteomics: Samples were lysed using glass beads with vortexing and digested using the modified FASP protocol

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Spin down and PBS wash after 15min of Ca2+ exposure

OD600 0.8

WT Calcium Control

FK506 Cyclosporin A

WT CC FK CA

4 0.8L cultures

1hr later add fraction to bring culture to 200mM CaCl2

Add 4mL of 1mg/mL FK506 or CylA in 90:10

EtOH:Tween 20

4mL of 90:10 EtOH:Tween 20

Metabolomics Workflow

Acquire Data

• Analyze metabolomics samples • Use accurate mass LC/MS to analyze samples

Analyze Data

• Mine data using MassHunter • Analyze in Mass Profiler Professional

Identify Compounds

• Identify metabolites using ID Browser • Search METLIN to annotate metabolites

Pathway Analysis

• Map results to pathways using Pathway Architect • Export protein list for next experiment

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TOF / Q-TOF For Discovery Metabolomics

Goal – Detect all metabolites Data is acquired in spectrum mode Metabolite tracking uses retention time and mass or mass fragments Statistical analysis is used to find differential metabolites (features) Feature identification is required for biological interpretation

Ideal for discovery metabolomics

Spectral quality • Accurate mass • Good mass resolution • 5 orders of dynamic range • High isotope ratio fidelity • Maintains performance at high

acquisition speeds • Sensitive

Compound identification • Accurate mass, isotope ratio • MS/MS with accurate mass, isotope

ratio (Q-TOF only)

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Agilent’s Complete Metabolomics Solution

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Agile

nt L

C/M

S an

d G

C/M

S

Q-TOF, TOF

Feature Finding Quantitate

MassHunter Qual

Alignment & Statistics

Statistics Visualization

Identify

ID Browser using Agilent Fiehn

Library, Agilent METLIN Library

(including lipids)

Pathway Analysis

Pathway Architect

Triple Quad, MSD

LC/MS and GC/MS Data can be analyzed together in the same project

Mass Profiler Professional

Separate & Detect

MassHunter Quant

or

Chromatographic Solutions for Separating Metabolites

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http://www.chem.agilent.com/Library/primers/Public/5990-4314EN.pdf

RP ANP GC/MS

LC/MS

Less Polar More Polar

LC/MS Analysis of Metabolites in Stressed Yeast

ESI (+) by RP

Polar compounds elute close to the void volume

ESI (+) by ANP

ANP separates polar compounds that were in the void volume with RP

Using both ANP and RP yields more comprehensive coverage

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7x10

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+ TIC Scan CA_RP_pos_1.d

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Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

6x10

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Cpd 593: 13.373: + ECC Scan CA_ANP_pos_1.d

Counts vs. Acquisition Time (min)1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14

Untargeted Feature Finding MassHunter Qual: Find by Molecular Feature Designed for accurate mass LC/TOF and LC/QTOF data

Find compound signals • Find co-eluting ions that are related

• Isotopes, adducts (such as Na+ / K+), and dimers

• Create a compound chromatogram and spectra

• Sum all ion signals into one value and report results as a “Feature” with retention time, neutral mass and volume

Fully automated processing Produces a CEF file for import into Mass Profiler Professional

Compound chromatogram

Compound spectra

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Mass Profiler Professional Statistical Analysis and Visualization Software

Designed primarily for MS data

• Also supports NMR

Performs many types of statistical analysis

• ANOVA, clustering, PCA, class prediction tools

ID Browser for compound identification

Export MS/MS target list

Pathway Architect for biological context

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PCA Plots for Yeast Metabolites Using Different Analytical Methodologies

RP Pos RP Neg APCI

ANP Pos ANP Neg

Culture Condition

Calcium Control

Cyclosporin A

FK506

Wild Type

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Increasing Your Confidence in Compound Identification

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Increasing confidence in identification

Compound identification data

Accurate Mass (AM)

AM +

Isotope Pattern

(IP)

AM +

Retention Time

(AMRT) + IP

MS/MS Library

MS/MS Library

+ AMRT

Confident compound identification is crucial for pathway visualization!

METLIN Personal Compound Database & Library Compound Identification

Q-TOF based LC-MS/MS library

Compounds in database - 64092

Compounds with MS/MS - 8040

• Collected in ESI +/- modes • Selected monoisotopic ion • Collected at three collision energies • Spectra reviewed for quality

• Fragment ions confirmed • Fragment ions mass corrected • Noise ions removed • Manually reviewed

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Most extensive database and MS/MS Library for metabolomics

Summary of Yeast Metabolomics Analyses Differential Features with METLIN Database Annotation

Number of Features After QC

METLIN Matches

p<0.05 Cutoff

METLIN Matches

RP-ESI pos 300 112 158 79

RP-ESI neg 523 141 418 115

RP-APCI pos 364 48 333 37

ANP-ESI pos 492 155 145 113

ANP-ESI neg 276 88 213 63

• ANP chromatography • ESI +/-

Polar metabolites

• RP chromatography • ESI +/- • APCI

Non-polar metabolites

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MS/MS Identification Using the Agilent METLIN Library

MS/MS spectra library matches:

• MS/MS spectra obtained at 10, 20 and 40eV collision energies

• Matched to METLIN PCDL library spectra

• Displayed as acquired spectra mirrored above library spectra

Hypoxanthine m/z 137.0458 (+) 10eV

20eV

40eV

83/99

56/99

63/99

Inosine m/z 267.0740 (-) 10eV

20eV

40eV

99/99

96/96

96/96

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Pathway Architect

Map and visualize data from one or two types of –omic data on pathways Search, browse and filter pathways Supports biological pathways from publicly available databases • WikiPathways • BioCyc

• Supported pathway formats • BioPAX 3 – Pathway Commons,

Reactome, NCI Nature Pathway • GPML – PathVisio –custom drawing

• Export compound list from pathways

Metabolite Data Overlay

List of all pathway entities, dynamically linked to pathway selection

Pathway Architect is an optional module in MassProfiler Professional

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Agilent-BridgeDB Resolving the Mapping Problem Between Databases

Solves the translation problem of identification names to pathway database names Automatic – does not require user intervention • Metabolites Identifiers

• KEGG, MetaCyc, PubChem. LMP, HMDB, ChEBI, and CAS

• Proteins Identifiers: • Swiss-Prot, UniProt, and UniProt/TrEMBL

• Genes Identifiers: • Entrez Gene, GenBank, Ensembl, EC

Number, RefSeq, UniGene, HUGO, HGNC, and EMBL

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One of the Stress Activated Pathways in Yeast

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Biology-directed Workflows to Targeted Proteomics

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Targeted Proteomics

Transforming target proteins to target peptides

Spectrum Mill and/or Skyline

List of target proteins

Pathway Architect

MassProfiler Professional

Metabolomics

Spectrum Mill

Discovery Proteomics

GeneSpring

Genomics Literature

Visualizing Discovery Proteomics Data in Pathways

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Q-TOF • Acquire data-dependent

proteomics data

Spectrum Mill • Protein database search • Produce protein summary • Export protein abundances

Mass Profiler Professional • Filter for quality data • Perform statistical analysis • Visualize differential results

Pathway Architect • Create a multi-omics project with

metabolomics results • Visualize proteomics and

metabolomics

Protein Discovery: Agilent 6550 Q-TOF with iFunnel Technologies Proteomics-optimized data-dependent acquisition on Q-TOFs

Outstanding sensitivity with iFunnel

High performance nano chromatography with Polaris-HR chip

New flexible G1992A nano source as alternative to HPLC-Chip interface

Excellent standard flow performance with AJS, 1290 and Advanced Bio column

25 Yeast Omics

Spectrum Mill – MPP Data Exchange Supporting a Label-free Proteomics Discovery Workflow

26 Yeast Omics

Spectrum Mill: Label-free Calculation of Protein Abundance Spectrum Mill provides this unique capability as part of protein database searching:

• Precursor area is extracted and stored

• MS/MS spectrum is matched to a peptide

• Peptide precursor area is assigned to that peptide

• Protein area = sum of areas for peptides matched to the protein

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Clear separation of yeast treatments

Quickly Map Discovery Proteomics Results to Pathways

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Spectrum Mill protein-protein comparison mode groups proteins across the entire data set

Export results to MPP

Pathway visualization in Pathway Architect

Combined Pathway Visualization of Discovery Proteomics and Metabolomics

metabolites proteins

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Biology-directed Workflows to Targeted Proteomics

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Targeted Proteomics

Transforming target proteins to target peptides

Spectrum Mill and/or Skyline

List of target proteins

Pathway Architect

MassProfiler Professional

Metabolomics

Spectrum Mill

Discovery Proteomics

GeneSpring

Genomics Literature

Creating Targeted Methods From Protein Lists: Metabolomics Proteomics

Metabolically interesting pathway

From discovery data • Use Spectrum Mill MRM Selector • Export peptide library to Skyline From in silico prediction • Copy protein accession number into Spectrum Mill and/or Skyline

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Targeted Proteomics: Using Pathway-Directed Information to Inform the Next Experiment

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Pathway Architect • Select perturbed pathway • Export protein accession numbers

• Visualize targeted results

Spectrum Mill and Skyline • Export results from Spectrum Mill • Predict peptides and MRMs for

new proteins • Refine using SRM Atlas

QQQ • Optimize best transitions • Perform DMRM analysis • Export areas to MPP

Mass Profiler Professional • Filter for quality data • Perform statistical analysis • Visualize differential results

Targeted Proteomics: Agilent 6490 QQQ with iFunnel Technologies Outstanding sensitivity with iFunnel

Excellent standard flow performance with AJS + 1290

Routine, robust nano LC with HPLC-Chip/QQQ

tMRM mode

Skyline workflow manuals

New Skyline Automation tool

33 Yeast Omics

Skyline – MPP Data Exchange Importing Targeted Results

34 Yeast Omics

Exporting Protein Areas From Skyline to MPP

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Review and process QQQ results in Skyline

Export results to MPP

Pathway visualization in Pathway Architect

Multi-omics Pathway Visualization of Metabolomic and Targeted Proteomics Results

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metabolites proteins

Pathway-based Multi-omic Discovery-to-Validation to Enable Integrated Biology

Pathway Architect

Skyline

Spectrum Mill

Pathway to PCDL

MPP

Q-TOF Acquisition

QQQ Acquisition

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Thank you!

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